Differentially expressed microrna molecules for the treatment and diagnosis of cancer

ABSTRACT

A significant challenge in cancer research field is to define molecular features that distinguish cancer stem cells from normal stem cells. In this study, microRNA (miRNA) expression profiles in human glioblastoma stem cells were compared to that of normal neural stem cells using combined microarray and deep sequencing analyses. These studies led to the identification of several miRNAs that are differentially expressed in glioblastoma stem cells and normal neural stem cells. Characterizing the role of these miRNAs in glioblastoma stem cells is important for the development of miRNA-based therapies that specifically target tumor stem cells, but spare normal stem cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/014,217, filed Aug. 29, 2013, now U.S. Pat. No. 9,315,809, issued onApr. 19, 2016, which claims the benefit of U.S. Provisional PatentApplication No. 61/694,698, filed Aug. 29, 2012 and now pending, whichis incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT INTEREST

The present invention was made with government support underR01-NS059546 and RC1-NS068370, each awarded by the National Institutesof Health and the National Institute of Neurological Disorders andStroke (NIH NINDS). The Government has certain rights in the invention.

BACKGROUND

MicroRNAs (miRNAs) are short 20-22 nucleotide RNA molecules that areexpressed in a tissue-specific and developmentally-regulated manner andfunction as negative regulators of gene expression in a variety ofeukaryotes. miRNAs are involved in numerous cellular processes includingdevelopment, proliferation, and differentiation [Ambros 2004; Bartel2004; Shi et al. 2010]. Increasing evidence has linked miRNAs to cancer[Esquela-Kerscher & Slack 2006] and are important regulators of many keypathways implicated in tumor pathogenesis [Asadi-Moghaddam et al. 2010],functioning as oncogenes or tumor suppressors in various tumors [Chenget al. 2010].

Although miRNAs have been shown to be differentially expressed invarious types of cancer cells as compared to normal cells, many cancersare thought to be maintained by a population of cancer stem cells thatretain stem cell properties, are highly tumorigenic, and displayincreased resistance to radiation and chemotherapy. As such, cancertherapies should target tumor stem cells, but spare normal stem cells.Therefore, there is a need for identifying miRNA molecules that aredifferentially expressed in tumor stem cell subpopulations as comparedto normal stem cells for the development of miRNA-based cancertherapeutics and diagnostics.

SUMMARY

One embodiment, methods for treating a cancer are provided. In certainembodiments, such methods include administering a therapeuticallyeffective amount of a pharmaceutical composition to a subject having thecancer, wherein the pharmaceutical composition comprises one or moretherapeutic agents which target one or more miRNA molecules that aredifferentially expressed in cancer stem cells as compared to normalcells. In other embodiments, such methods include contacting a cancercell or cancer stem cell with one or more miRNA molecules that have orimpart at least one tumor suppressor activity.

In another embodiment, methods for diagnosing a cancer are provided.Such methods include detecting a test level of one or more miRNAmolecules in a biological sample from a subject; comparing the testlevel to a reference level; and diagnosing the subject as having thecancer when the test level is significantly different than the referencelevel.

In another embodiment, an miRNA expression signature is provided that isspecific to a cancer stem cell. The miRNA expression signature includesone or more miRNA molecules that are differentially expressed in thecancer stem cell.

According to the embodiments described above, the one or more miRNAmolecules may be selected from miR-10a, miR-10b, miR-140-3p, miR-140-5p,miR-204, miR-424, miR-34a, miR-193a-3p, miR-455-5p, miR-455-3p, miR-9,miR-10a, miR-148a, miR-488, miR-196a1, miR-182, miR-96, miR-193b,miR-27a, miR-196b, miR-10b, miR-29b2, miR-23a, miR-107, miR-542-3p,miR-93, miR-365a4, miR-450a, miR-100, miR-105, miR-363, miR-105,miR-106b, miR-15b, miR-21, miR-376c, miR-93, miR-99b, miR-155, miR-33a,miR-876-3p, miR-362-3p, miR-25, let-7i, miR-423-3p, miR-34b, miR-16-2,-miR-29a, miR-30d, miR-320, miR-181c, miR-128a, miR-21, let-7d,miR-450b-5p, miR-371-5p, miR-1245, miR-335, miR-492, miR-874, miR-30b,miR-193a-5p, miR-602, miR-346, miR-663, miR-25, miR-219-5p6, miR-184,miR-135a7, miR-584, miR-665, miR-638, miR-503, miR-628-3p, miR-381,miR-78, miR-92b, miR-149, miR-135b, miR-302d, miR-498, miR-766,miR-1389, miR-623, miR-519c-5p, miR-182, miR-494, miR-129-5p10,miR-513-5p, miR-200b, miR-634, miR-654-5p, miR-518b, miR-658, miR-373,miR-30c-2, miR-130a, miR-557, miR-551a, miR-637, miR-518c, miR-525-5p,miR-596, miR-552, miR-625, miR-183, miR-187, miR-544, miR-891a,miR-519e, miR-933, miR-939, miR-214, miR-671-5p, miR-137, miR-92b,miR-525-3p, miR-19a, and miR-409-5p.

The embodiments described above may be specific to and may be used totreat or diagnose any applicable cancer including, but not limited to,bone cancer, bladder cancer, brain cancer, breast cancer, cancer of theurinary tract, carcinoma, cervical cancer, colon cancer, esophagealcancer, gastric cancer, head and neck cancer, hepatocellular cancer,liver cancer, lung cancer, lymphoma and leukemia, melanoma, ovariancancer, pancreatic cancer, pituitary cancer, prostate cancer, rectalcancer, renal cancer, sarcoma, testicular cancer, thyroid cancer, anduterine cancer. In one embodiment, the cancer is brain cancer, inparticular, glioblastoma.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show the morphology, differentiation and a growth curve ofglioblastoma stem cells (GSCs) and neural stem cells (NSCs). FIG. 1Ashows representative images of neurospheres from normal human neuralstem cell lines 1-3 (NSC1-3) and glioblastoma stem cell lines 1-3(GSC1-3). FIG. 1B illustrates the multipotency of NSCs and GSCs. Wheninduced into differentiation, both NSCs and GSCs gave rise to Tuj1+neurons (green) and GFAP+ astrocytes (red). Representative images ofNSC1 and GSC1 differentiation were shown. Nuclear Dapi staining wasshown in blue. FIG. 1C. H&E staining of coronal sections fromGSC-transplanted brains. The tumor region was indicated by an arrow,shown in dark purple color.

FIGS. 2A-2E illustrate the real-time RT-PCR validation of miRNAexpression in glioblastoma stem cells. The expression levels of miR-10a(FIG. 2A), miR-10b (FIG. 2B), miR-140-5p (FIG. 2C), miR-124 (FIG. 2D),and miR-874 (FIG. 2E) in three glioblastoma stem cell (GSC) lines weremeasured by real-time RT-PCR, and compared to their expression in threeneural stem cell (NSC) lines. The expression shown in each cell line isrelative to the expression in NSC1, with the expression in NSC1 as 1.Error bars are standard deviation of the mean. * p<0.001, ** p<0.005 byone way ANOVA test.

FIGS. 3A-3D illustrate the real-time RT-PCR analysis of miRNA expressionin glioblastoma tissues. The expression levels of miR-10a (FIG. 3A),miR-10b (FIG. 3B), miR-124 (FIG. 3C), and miR-874 (FIG. 3D) in 9glioblastoma tissues and 4 normal brain tissues were determine byreal-time RT-PCR analysis, shown in scatted graph and bar graph. Errorbars are standard error of the mean. p value was obtained by student'st-test.

FIGS. 4A-4F illustrate the expression of miR-10b targets in glioblastomastem cells. FIGS. 4A and 4B show the base-pairing of hsa-miR-10a (SEQ IDNO: 1) and hsa-miR-10b (SEQ ID NO: 6) with the 3′ UTR of CSMD1 gene (SEQID NOS: 2-5). FIG. 4C illustrates miR-10a-mediated repression ofluciferase reporter gene downstream of 3′ UTR of CSMD1. Luciferasereporter gene under the control of wild type (WT) or mutant (MT) CSMD13′ UTR was transfected into HEK 293 cells along with control, miR-10aRNA duplexes, or the combination of miR-10a RNA duplexes and a miR-10ainhibitor. * p<0.001 by student's t-test. FIG. 4D illustratesmiR-10b-mediated repression of luciferase reporter gene downstream of 3′UTR of CSMD1. WT or MT CSMD1 3′ UTR luciferase reporter was transfectedinto HEK 293 cells along with control, miR-10b RNA duplexes, or thecombination of miR-10b RNA duplexes and a miR-10b inhibitor. * p<0.005by student's t-test. FIG. 4E illustrates expression of CSMD1 inglioblastoma stem cell line 1 (GSC) and neural stem cell line 1 (NSC)determined by real-time RT-PCR analysis. FIG. 4F illustrates expressionof HOXD10 in GSC and NSC determined by real-time RT-PCR analysis. Forall panels, data shown are mean±standard deviation of three replicates.** p<0.01 by student's t-test for both panels E and F. The term “hsa” infront of the miRNA indicates the species homo sapiens.

FIGS. 5A-5H show that miR-124 targets NRAS and PIM3 expression. FIG. 5Aillustrates the base-pairing of hsa-miR-124 (SEQ ID NO: 7) with the 3′UTR of NRAS gene (SEQ ID NO: 8-10). FIG. 5B illustrates themiR-124-mediated repression of luciferase reporter gene downstream of 3′UTR of NRAS. Luciferase reporter gene under the control of wild type(WT) or mutant (MT) NRAS 3′ UTR was transfected into HEK 293 cells alongwith control, miR-124 RNA duplexes, or the combination of miR-124 RNAduplexes and a miR-124 inhibitor. *p<0.001 by student's t-test. FIG. 5Cillustrates Western blot analysis of NRAS expression in control RNA,miR-124 RNA duplexes, or the combination of miR-124 RNA duplexes and amiR-124 inhibitor-transfected GSC1 cells. FIG. 5D illustrates expressionof NRAS in GSC1 and NSC1 determined by real-time RT-PCR analysis.*p<0.05 by student's t-test. FIG. 5E shows the base-pairing ofhsa-miR-124 (SEQ ID NO: 7) with the 3′ UTR of PIM3 gene (SEQ ID NO:11-12). FIG. 5F illustrates miR-124-mediated repression of luciferasereporter gene downstream of 3′ UTR of PIM3. Luciferase reporter geneunder the control of wild type (WT) or mutant (MT) PIM3 3′ UTR wastransfected into HEK 293 cells along with control, miR-124 RNA duplexes,or the combination of miR-124 RNA duplexes and a miR-124 inhibitor.*p<0.001 by student's t-test. FIG. 5G illustrates Western blot analysisof PIM3 expression in control RNA, miR-124 RNA duplexes, or thecombination of miR-124 RNA duplexes and a miR-124 inhibitor-transfectedGSC1 cells. FIG. 5H illustrates the expression of PIM3 in GSC1 and NSC1determined by real-time RT-PCR analysis. **p<0.001 by student's t-test.For all panels, data shown are mean±standard deviation of threereplicates.

FIGS. 6A-6B show pathways targeted by deregulated miRNAs in glioblastomastem cells. Common miRNA targets were subjected to DAVID functionalannotation with KEGG pathway analysis. FIG. 6A illustrates that theup-regulated miRNAs in glioblastoma stem cells were predicted to targetthe p53 pathway. The p53-centered pathway has been shown to regulatecell cycle, apoptosis, angiogenesis, metastasis, and genome stability.FIG. 6B illustrates that the down-regulated miRNAs were predicted totarget components of the IGF pathway. Various components of the IGFsignaling pathways were targeted by down-regulated miRNAs. The IGFpathway has been shown to enhance cell growth, survival, and migration.

FIGS. 7A-7D illustrate viral transduction of miR-874 in glioblastomalines PBT017 and PBT707 (FIGS. 7A and 7C, respectively), and resultingreduced growth of the PBT017 and PBT707 glioblastoma cells due toexpression of miR-874 (FIGS. 7B and 7D, respectively).

DETAILED DESCRIPTION

Methods for diagnosing and treating cancer using one or more miRNAmolecules are provided herein. Such methods may be used to treat ordiagnose any cancer or tumor cell type including bone cancer, bladdercancer, brain cancer, breast cancer, cancer of the urinary tract,carcinoma, cervical cancer, colon cancer, esophageal cancer, gastriccancer, head and neck cancer, hepatocellular cancer, liver cancer, lungcancer, lymphoma and leukemia, melanoma, ovarian cancer, pancreaticcancer, pituitary cancer, prostate cancer, rectal cancer, renal cancer,sarcoma, testicular cancer, thyroid cancer, and uterine cancer. Inaddition, the methods may be used to treat tumors that are malignant(e.g., primary or metastatic cancers) or benign (e.g., hyperplasia,cyst, pseudocyst, hematoma, and benign neoplasm).

In one embodiment, the methods described herein may be used to treat ordiagnose a brain cancer including, but not limited to, gliomas (e.g.,astrocytomas, oligodiendrogliomas, mixed gliomas, enendymomas, brainstem gliomas) meningiomas, pineal gland and pituitary gland tumors,primary central nervous system lymphomas, medulloblastomas,craniopharyngiomas and acoustic neuromas.

Astrocytomas, the most common type of glioma, have been subclassifiedinto four subtypes (“or grades”) using the World Health Organization(WHO) classification system for tumor identification [Louis et al.2007]. This grading scheme represents a malignancy scale and is animportant factor influencing the choice of therapies. According to theWHO, a grade I astrocytoma is classified as a pilocytic astrocytoma, agrade II astrocytoma is classified as a diffuse astrocytoma, a grade IIIastrocytoma is classified as an anaplastic astrocytoma, and a grade IVastrocytoma is classified as a glioblastoma—the most malignant grade[Louis et al. 2007]. Glioblastoma is the most common and aggressiveprimary brain tumor with median survival time of 14 months afterdiagnosis [Louis et al. 2007]. Currently, no effective treatment hasbeen developed for glioblastoma patients. Therefore, although the miRNAmolecules used according to the methods described herein may be used totreat or diagnose any type of cancer, the Examples are primarilydirected to the identification of miRNA molecules related toglioblastoma.

Recent studies have suggested that glioblastomas are maintained by asmall population of cancer stem cells that retain stem cell properties,are highly tumorigenic, and display increased resistance to radiationand chemotherapy [Singh et al. 2004; Bao et al. 2006; Godlewski et al.2010]. These treatment-resistant tumor cell subpopulations are the cellpopulations that effective therapies must target [Godlewski et al.2010].

miRNAs have been shown to be differentially expressed in glioblastomatissues compared to normal brain tissues. For example, miRNA 21 isoverexpressed in glioblastoma tissues, relative to surrounding normalbrain tissues [Conti et al. 2009]. miR-26a is also amplified inglioblastoma tissues. By targeting the tumor suppressor Pten,overexpression of miR-26a facilitates tumorigenesis and predicts a poorsurvival [Huse et al. 2009; Kim et al. 2010]. On the other hand,miR-124, miR-137 and miR-451 exhibit reduced expression in malignantglioblastoma tissues relative to normal brain tissues [Silber et al.2008; Gal et al. 2008]. The expression of these miRNAs is also reducedin glioblastoma stem cells relative to bulk tumor cells. Overexpressionof these miRNAs in glioblastoma stem cells inhibits cell proliferationand induces neural differentiation, suggesting a tumor suppressor rolefor these miRNAs. These studies suggest that some miRNAs may be used astherapeutic agents for targeting glioblastoma stem cells. However, braintumor stem cells share a core developmental program with normal neuralstem cells [Cheng et al. 2010]. Optimal therapies should be designed totarget tumor stem cells, but spare normal stem cells. Therefore,identifying miRNAs that are differentially expressed in glioblastomastem cells and normal neural stem cells is important for the developmentof optimal miRNA-based therapies and diagnostics for glioblastomapatients.

Therefore, in some embodiments, methods for treating cancer (e.g.,glioblastoma) include a step of administering a therapeuticallyeffective amount of a pharmaceutical composition that includes one ormore therapeutic agents which target and/or affect the expression levelof one or more miRNA molecules that are differentially expressed incancer stem cells (e.g., glioblastoma stem cells) and/or other cancercells as compared to healthy cells or healthy stem cells. A therapeuticagent which targets a target molecule (e.g., a target miRNA moleculethat is differentially expressed) means that said therapeutic agent,when administered to a subject or is otherwise exposed to the targetmolecule, results in an alteration in the expression or activity of thetarget molecule. Such alteration may include, but is not limited to,inhibition, suppression activation, agonization, or otherwise cause achange in the expression or activity level of the target molecule. Thetherapeutic agent may directly cause such a change, or may act onupstream or downstream targets that ultimately result in a change to thetarget molecule.

According to some embodiments, the one or more miRNA molecules that aretargeted may include, but are not limited to, miR-10a, miR-10b,miR-140-3p, miR-140-5p, miR-204, miR-424, miR-34a, miR-193a-3p,miR-455-5p, miR-455-3p, miR-9, miR-10a, miR-148a, miR-488, miR-196a1,miR-182, miR-96, miR-193b, miR-27a, miR-196b, miR-10b, miR-29b2,miR-23a, miR-107, miR-542-3p, miR-93, miR-365a4, miR-450a, miR-100,miR-105, miR-363, miR-105, miR-106b, miR-15b, miR-21, miR-376c, miR-93,miR-99b, miR-155, miR-33a, miR-876-3p, miR-362-3p, miR-25, let-7i,miR-423-3p, miR-34b, miR-16-2, -miR-29a, miR-30d, miR-320, miR-181c,miR-128a, miR-21, let-7d, miR-450b-5p, miR-371-5p, miR-1245, miR-335,miR-492, miR-874, miR-30b, miR-193a-5p, miR-602, miR-346, miR-663,miR-25, miR-219-5p6, miR-184, miR-135a7, miR-584, miR-665, miR-638,miR-503, miR-628-3p, miR-381, miR-78, miR-92b, miR-149, miR-135b,miR-302d, miR-498, miR-766, miR-1389, miR-623, miR-519c-5p, miR-182,miR-494, miR-129-5p10, miR-513-5p, miR-200b, miR-634, miR-654-5p,miR-518b, miR-658, miR-373, miR-30c-2, miR-130a, miR-557, miR-551a,miR-637, miR-518c, miR-525-5p, miR-596, miR-552, miR-625, miR-183,miR-187, miR-544, miR-891a, miR-519e, miR-933, miR-939, miR-214,miR-671-5p, miR-137, miR-92b, miR-525-3p, miR-19a, and miR-409-5p.

In one embodiment, the one or more therapeutic agents target one or moremiRNA molecules that are significantly upregulated in cancer stem cells(e.g., glioblastoma stem cells) and/or other cancer cells as compared tohealthy cells or healthy stem cells. The one or more miRNA moleculesthat are significantly upregulated include, but are not limited to,miR-10a, miR-10b, miR-140-3p, miR-140-5p, miR-204, miR-424, miR-34a,miR-193a-3p, miR-455-5p, miR-455-3p, miR-9, miR-10a, miR-148a, miR-488,miR-196a1, miR-182, miR-96, miR-193b, miR-27a, miR-196b, miR-10b,miR-29b2, miR-23a, miR-107, miR-542-3p, miR-93, miR-365a4, miR-450a,miR-100, miR-105, miR-363, miR-105, miR-106b, miR-15b, miR-21, miR-376c,miR-93, miR-99b, miR-155, miR-33a, miR-876-3p, miR-362-3p, miR-25,let-7i, miR-423-3p, miR-34b, miR-16-2, -miR-29a, miR-30d, miR-320,miR-181c, miR-128a, miR-21, let-7d, and miR-450b-5p. In one embodiment,the one or more miRNA molecules that are significantly upregulatedinclude, but are not limited to miR-10a, miR-10b, miR-140-3p,miR-140-5p, miR-204, miR-424, miR-455-5p, miR-455-3p.

In another embodiment, the one or more therapeutic agents target one ormore miRNA molecules that are significantly downregulated in cancer stemcells (e.g., glioblastoma stem cells) and/or other cancer cells ascompared to healthy cells or healthy stem cells. The one or more miRNAmolecules that are significantly downregulated include, but are notlimited to, miR-371-5p, miR-1245, miR-335, miR-492, miR-874, miR-30b,miR-193a-5p, miR-602, miR-346, miR-663, miR-25, miR-219-5p6, miR-184,miR-135a7, miR-584, miR-665, miR-638, miR-503, miR-628-3p, miR-381,miR-78, miR-92b, miR-149, miR-135b, miR-302d, miR-498, miR-766,miR-1389, miR-623, miR-519c-5p, miR-182, miR-494, miR-129-5p10,miR-513-5p, miR-200b, miR-634, miR-654-5p, miR-518b, miR-658, miR-373,miR-30c-2, miR-130a, miR-557, miR-551a, miR-637, miR-518c, miR-525-5p,miR-596, miR-552, miR-625, miR-183, miR-187, miR-544, miR-891a,miR-519e, miR-933, miR-939, miR-214, miR-671-5p, miR-137, miR-92b,miR-525-3p, miR-19a, and miR-409-5p. In one embodiment, the one or moremiRNA molecules that are significantly downregulated include, but arenot limited to hsa-miR-371-5p, hsa-miR-124-1, hsa-miR-124-2,hsa-miR-124-3, hsa-miR-335, hsa-miR-492, hsa-miR-874, hsa-miR-30b, andhsa-miR-602.

In certain embodiments, treating cancer (e.g., glioblastoma) in asubject having the cancer may be accomplished by contacting a cancercell (e.g., glioblastoma cell) or cancer stem cell (e.g., glioblastomastem cell) with one or more miRNA molecules that have or impart at leastone tumor suppressor activity. In such embodiments, the one or moremiRNA molecules act as a therapeutic agent directly. Tumor suppressoractivities may include, but are not limited to, suppression orinhibition of cell growth and/or cell division. In some examples, anmiRNA that is found to be significantly down-regulated in cancer cellsor cancer stem cells relative to normal stem cells or normal cells mayact as a tumor suppressor. Thus, miRNA molecules that have or imparttumor suppressor activities may include, but are not limited tomiR-371-5p, miR-1245, miR-335, miR-492, miR-874, miR-30b, miR-193a-5p,miR-602, miR-346, miR-663, miR-25, miR-219-5p6, miR-184, miR-135a7,miR-584, miR-665, miR-638, miR-503, miR-628-3p, miR-381, miR-78,miR-92b, miR-149, miR-135b, miR-302d, miR-498, miR-766, miR-1389,miR-623, miR-519c-5p, miR-182, miR-494, miR-129-5p10, miR-513-5p,miR-200b, miR-634, miR-654-5p, miR-518b, miR-658, miR-373, miR-30c-2,miR-130a, miR-557, miR-551a, miR-637, miR-518c, miR-525-5p, miR-596,miR-552, miR-625, miR-183, miR-187, miR-544, miR-891a, miR-519e,miR-933, miR-939, miR-214, miR-671-5p, miR-137, miR-92b, miR-525-3p,miR-19a, and miR-409-5p. In one embodiment, the one or more miRNAmolecules that have or impart tumor suppressor activities may include,but are not limited to, hsa-miR-371-5p, hsa-miR-124-1, hsa-miR-124-2,hsa-miR-124-3, hsa-miR-335, hsa-miR-492, hsa-miR-874, hsa-miR-30b, andhsa-miR-602.

In certain embodiments, the one or more therapeutic agents target atleast one miRNA molecules that is significantly upregulated and at leastone miRNA molecule that is significantly downregulated in cancer stemcells (e.g., glioblastoma stem cells) and/or other cancer cells ascompared to healthy cells or healthy stem cells. The one or more miRNAmolecules that are significantly upregulated include, but are notlimited to miR-10a, miR-10b, miR-140-3p, miR-140-5p, miR-204, miR-424,miR-455-5p, miR-455-3p; and the one or more miRNA molecules that aresignificantly downregulated include, but are not limited tohsa-miR-371-5p, hsa-miR-124-1, hsa-miR-124-2, hsa-miR-124-3,hsa-miR-335, hsa-miR-492, hsa-miR-874, hsa-miR-30b, and hsa-miR-602.

Therapeutic agents that may be used in accordance with the methodsdescribed herein may include, but are not limited to, (i) miRNAinhibitors to inhibit or silence specific miRNA molecules that areupregulated in glioblastoma or other cancers; (ii) miRNA inhibitors tosequester endogenous miRNA, thereby blocking the endogenous miRNAfunction (i.e., the “sponge method,” see Ebert et al. 2007, which ishereby incorporated by reference as if fully set forth herein); and(iii) agents that increase the level of specific miRNA molecules, forexample, an miRNA that has been identified as a downregulated miRNAmolecule to replace the same miRNA that has a lower expression (such asthose described above) or an miRNA expression vector that causes a cellto overexpresses such a downregulated miRNA molecule; and (iv) miRNAmolecules that can directly replace a downregulated miRNA molecule.

Because the sequences of many miRNA molecules have been previouslydescribed (see http://www.mirbase.org/), inhibitors specific for one ormore upregulated miRNA molecules, such as those described above, may beobtained commercially (e.g., from Thermo Scientific Dharmacon), or maybe developed based on designing microRNA hairpin inhibitors, antisenseinhibitors (e.g., 2′-O-methyl miRNA antisense RNAs), LNA miRNAinhibitors, RNA interference molecules (e.g., shRNA, sRNA), aptamers, orother suitable complementary miRNA inhibitors.

Delivery of the therapeutic agents described above may be accomplishedby any suitable method including, but not limited to, viralvector-delivery (e.g., lentiviral vector delivery, AAV-viral vectordelivery, adenoviral vector delivery), dendrimer-mediated delivery,nanoparticle-mediated delivery or a combination thereof (e.g.,dendrimer-based nanoparticle delivery).

In addition to the therapeutic agents, the pharmaceutical compositionmay also include a pharmaceutical carrier. A “pharmaceuticallyacceptable carrier” refers to a pharmaceutically acceptable material,composition, or vehicle that is involved in carrying or transporting acompound of interest from one tissue, organ, or portion of the body toanother tissue, organ, or portion of the body. For example, the carriermay be a liquid or solid filler, diluent, excipient, solvent, orencapsulating material, or some combination thereof. Each component ofthe carrier must be “pharmaceutically acceptable” in that it must becompatible with the other ingredients of the formulation. It also mustbe suitable for contact with any tissue, organ, or portion of the bodythat it may encounter, meaning that it must not carry a risk oftoxicity, irritation, allergic response, immunogenicity, or any othercomplication that outweighs its therapeutic benefits.

The pharmaceutical composition that may be used in accordance with themethods described herein may be administered, by any suitable route ofadministration, alone or as part of a pharmaceutical composition. Aroute of administration may refer to any administration pathway known inthe art, including but not limited to aerosol, enteral, nasal,ophthalmic, oral, parenteral, rectal, transdermal (e.g., topical creamor ointment, patch), or vaginal. “Transdermal” administration may beaccomplished using a topical cream or ointment or by means of atransdermal patch. “Parenteral” refers to a route of administration thatis generally associated with injection, including infraorbital,infusion, intraarterial, intracapsular, intracardiac, intradermal,intramuscular, intraperitoneal, intrapulmonary, intraspinal,intrasternal, intrathecal, intrauterine, intravenous, subarachnoid,subcapsular, subcutaneous, transmucosal, or transtracheal.

The term “effective amount” as used herein refers to an amount of acompound that produces a desired effect. For example, a population ofcells may be contacted with an effective amount of a compound to studyits effect in vitro (e.g., cell culture) or to produce a desiredtherapeutic effect ex vivo or in vitro. An effective amount of acompound may be used to produce a therapeutic effect in a subject, suchas preventing or treating a target condition, alleviating symptomsassociated with the condition, or producing a desired physiologicaleffect. In such a case, the effective amount of a compound is a“therapeutically effective amount,” “therapeutically effectiveconcentration” or “therapeutically effective dose.” The preciseeffective amount or therapeutically effective amount is an amount of thecomposition that will yield the most effective results in terms ofefficacy of treatment in a given subject or population of cells. Thisamount will vary depending upon a variety of factors, including but notlimited to the characteristics of the compound (including activity,pharmacokinetics, pharmacodynamics, and bioavailability), thephysiological condition of the subject (including age, sex, disease typeand stage, general physical condition, responsiveness to a given dosage,and type of medication) or cells, the nature of the pharmaceuticallyacceptable carrier or carriers in the formulation, and the route ofadministration. Further an effective or therapeutically effective amountmay vary depending on whether the compound is administered alone or incombination with another compound, drug, therapy or other therapeuticmethod or modality. One skilled in the clinical and pharmacological artswill be able to determine an effective amount or therapeuticallyeffective amount through routine experimentation, namely by monitoring acell's or subject's response to administration of a compound andadjusting the dosage accordingly. For additional guidance, seeRemington: The Science and Practice of Pharmacy, 21^(st) Edition, Univ.of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins,Philadelphia, Pa., 2005, which is hereby incorporated by reference as iffully set forth herein.

“Treating” or “treatment” of a condition may refer to preventing thecondition, slowing the onset or rate of development of the condition,reducing the risk of developing the condition, preventing or delayingthe development of symptoms associated with the condition, reducing orending symptoms associated with the condition, generating a complete orpartial regression of the condition, or some combination thereof.Treatment may also mean a prophylactic or preventative treatment of acondition.

In some embodiments, an miRNA molecule or other biomarker that is either(i) upregulated or overexpressed; or (ii) downregulated orunderexpressed can also be referred to as being “differentiallyexpressed” as compared to a “normal” expression level or value of themiRNA molecule or other biomarker that indicates or is a sign of anormal process or an absence of a disease or other condition in anindividual. Thus, “differential expression” of an miRNA molecule orother biomarker can also be referred to as a variation from a “normal”expression level of the biomarker. Differential expression includesquantitative, as well as qualitative, differences in the temporal orcellular expression pattern in a gene or its expression products among,for example, normal and diseased cells (e.g., normal stem cells vs.cancer stem cells; normal cells vs. cancer cells, or a combinationthereof), or among cells which have undergone different disease eventsor disease stages.

Further, the phrase “differentially expressed” refers to a difference inthe quantity or intensity of a marker (e.g., miRNA) present in abiological sample taken from subjects having a cancer as compared to acomparable sample taken from subjects who do not have the cancer. Forexample, an miRNA molecule is differentially expressed between thesamples if the amount of the miRNA molecule in one sample issignificantly different (i.e., p<0.05) from the amount of the miRNAmolecule in the other sample. It should be noted that if the miRNAmolecule or other marker is detectable in one sample and not detectablein the other, then the miRNA molecule can be considered to bedifferentially present.

The identification of the miRNAs described above and in the Examplesbelow suggests that these miRNAs may be used as novel diagnosticmarkers. Thus, according to some embodiments, methods for diagnosingcancer (e.g., glioblastoma) include a step of detecting a test level ofone or more miRNA molecules in a biological sample from a subject who issuspected of having the cancer.

According to some embodiments, the one or more miRNA molecules that aredetected may include, but are not limited to, miR-10a, miR-10b,miR-140-3p, miR-140-5p, miR-204, miR-424, miR-34a, miR-193a-3p,miR-455-5p, miR-455-3p, miR-9, miR-10a, miR-148a, miR-488, miR-196a1,miR-182, miR-96, miR-193b, miR-27a, miR-196b, miR-10b, miR-29b2,miR-23a, miR-107, miR-542-3p, miR-93, miR-365a4, miR-450a, miR-100,miR-105, miR-363, miR-105, miR-106b, miR-15b, miR-21, miR-376c, miR-93,miR-99b, miR-155, miR-33a, miR-876-3p, miR-362-3p, miR-25, let-7i,miR-423-3p, miR-34b, miR-16-2, -miR-29a, miR-30d, miR-320, miR-181c,miR-128a, miR-21, let-7d, miR-450b-5p, miR-371-5p, miR-1245, miR-335,miR-492, miR-874, miR-30b, miR-193a-5p, miR-602, miR-346, miR-663,miR-25, miR-219-5p6, miR-184, miR-135a7, miR-584, miR-665, miR-638,miR-503, miR-628-3p, miR-381, miR-78, miR-92b, miR-149, miR-135b,miR-302d, miR-498, miR-766, miR-1389, miR-623, miR-519c-5p, miR-182,miR-494, miR-129-5p10, miR-513-5p, miR-200b, miR-634, miR-654-5p,miR-518b, miR-658, miR-373, miR-30c-2, miR-130a, miR-557, miR-551a,miR-637, miR-518c, miR-525-5p, miR-596, miR-552, miR-625, miR-183,miR-187, miR-544, miR-891a, miR-519e, miR-933, miR-939, miR-214,miR-671-5p, miR-137, miR-92b, miR-525-3p, miR-19a, and miR-409-5p.

In one embodiment, the one or more miRNA molecules are significantlyupregulated in cancer stem cells (e.g., glioblastoma stem cells) and/orother cancer cells as compared to healthy cells or healthy stem cells.The one or more miRNA molecules that are significantly upregulatedinclude, but are not limited to, miR-10a, miR-10b, miR-140-3p,miR-140-5p, miR-204, miR-424, miR-34a, miR-193a-3p, miR-455-5p,miR-455-3p, miR-9, miR-10a, miR-148a, miR-488, miR-196a1, miR-182,miR-96, miR-193b, miR-27a, miR-196b, miR-10b, miR-29b2, miR-23a,miR-107, miR-542-3p, miR-93, miR-365a4, miR-450a, miR-100, miR-105,miR-363, miR-105, miR-106b, miR-15b, miR-21, miR-376c, miR-93, miR-99b,miR-155, miR-33a, miR-876-3p, miR-362-3p, miR-25, let-7i, miR-423-3p,miR-34b, miR-16-2, -miR-29a, miR-30d, miR-320, miR-181c, miR-128a,miR-21, let-7d, and miR-450b-5p. In one embodiment, the one or moremiRNA molecules that are significantly upregulated include, but are notlimited to miR-10a, miR-10b, miR-140-3p, miR-140-5p, miR-204, miR-424,miR-455-5p, miR-455-3p.

In another embodiment, the one or more miRNA molecules are significantlydownregulated in cancer stem cells (e.g., glioblastoma stem cells)and/or other cancer cells as compared to healthy cells or healthy stemcells. The one or more miRNA molecules that are significantlydownregulated include, but are not limited to, miR-371-5p, miR-1245,miR-335, miR-492, miR-874, miR-30b, miR-193a-5p, miR-602, miR-346,miR-663, miR-25, miR-219-5p6, miR-184, miR-135a7, miR-584, miR-665,miR-638, miR-503, miR-628-3p, miR-381, miR-78, miR-92b, miR-149,miR-135b, miR-302d, miR-498, miR-766, miR-1389, miR-623, miR-519c-5p,miR-182, miR-494, miR-129-5p10, miR-513-5p, miR-200b, miR-634,miR-654-5p, miR-518b, miR-658, miR-373, miR-30c-2, miR-130a, miR-557,miR-551a, miR-637, miR-518c, miR-525-5p, miR-596, miR-552, miR-625,miR-183, miR-187, miR-544, miR-891a, miR-519e, miR-933, miR-939,miR-214, miR-671-5p, miR-137, miR-92b, miR-525-3p, miR-19a, andmiR-409-5p. In one embodiment, the one or more miRNA molecules that aresignificantly downregulated include, but are not limited tohsa-miR-371-5p, hsa-miR-124-1, hsa-miR-124-2, hsa-miR-124-3,hsa-miR-335, hsa-miR-492, hsa-miR-874, hsa-miR-30b, and hsa-miR-602.

In certain embodiments, the one or more miRNA molecules may include atleast one miRNA molecule that is significantly upregulated and at leastone miRNA molecule that is significantly downregulated in cancer stemcells (e.g., glioblastoma stem cells) and/or other cancer cells ascompared to healthy cells or healthy stem cells. The one or more miRNAmolecules that are significantly upregulated include, but are notlimited to miR-10a, miR-10b, miR-140-3p, miR-140-5p, miR-204, miR-424,miR-455-5p, miR-455-3p; and the one or more miRNA molecules that aresignificantly downregulated include, but are not limited tohsa-miR-371-5p, hsa-miR-124-1, hsa-miR-124-2, hsa-miR-124-3,hsa-miR-335, hsa-miR-492, hsa-miR-874, hsa-miR-30b, and hsa-miR-602.

According to the embodiments described herein, a biological sample mayrefer to any material, biological fluid, tissue, or cell obtained orotherwise derived from an individual including, but not limited to,blood (including whole blood, leukocytes, peripheral blood mononuclearcells, buffy coat, plasma, and serum), sputum, tears, mucus, nasalwashes, nasal aspirate, breath, urine, semen, saliva, meningeal fluid,amniotic fluid, glandular fluid, lymph fluid, milk, bronchial aspirate,synovial fluid, joint aspirate, cells, a cellular extract, andcerebrospinal fluid. This also includes experimentally separatedfractions of all of the preceding. For example, a blood sample can befractionated into serum or into fractions containing particular types ofblood cells, such as red blood cells or white blood cells (leukocytes).If desired, a sample can be a combination of samples from an individual,such as a combination of a tissue and fluid sample. A biological samplemay also include a biological tissue sample, such as a sample obtainedby tissue biopsy or by surgical excision. A biological sample may alsoinclude materials derived from a tissue culture or a cell culture.Further, a biological sample may be derived by taking biological samplesfrom a number of individuals and pooling them or pooling an aliquot ofeach individual's biological sample. The pooled sample can be treated asa sample from a single individual and if the presence of cancer isestablished in the pooled sample, then each individual biological samplecan be re-tested to determine which individuals have cancer.

A test level of an miRNA molecule or other biomarker refers to an amountof a biomarker, such as an miRNA molecule, in a subject's undiagnosedbiological sample. The test level may be compared to that of a controlsample, or may be analyzed based on a reference or control level thathas been previously established to determine a status of the sample.Such a status may be a diagnosis, prognosis or evaluation of a diseaseor condition. In one embodiment, the disease is cancer. A test sample ortest amount can be either in absolute amount (e.g., nanogram/mL ormicrogram/mL) or a relative amount (e.g., relative intensity ofsignals).

The test level of the one or more miRNA molecules may be detected by anysuitable method of measurement or quantification known in the artincluding, but not limited to, reverse transcriptase-polymerase chainreaction (RT-PCR) methods (including quantitative and qualitative RT-PCRmethods), microarray, serial analysis of gene expression (SAGE), geneexpression analysis by massively parallel signature sequencing (MPSS) ordeep sequencing methods such as those described below, immunoassays suchas ELISA, immunohistochemistry (IHC), mass spectrometry (MS) methods,transcriptomics and proteomics. Several of the detection methodsdescribed above include a transformative step, wherein the one or moremiRNA molecules that are present in a biological sample are converted ortransformed into structural, visual or other tangible manifestation ofthe amount of miRNA (e.g., a cDNA molecule, a reporter signal, afluorescent, luminescent or radioactive signal, a labeled antibody, agraph, a tracing).

In some embodiments, the test level of the one or more miRNA moleculesmay be detected using a set of reagents which contain miRNA detectionagents specific to a set of one or more miRNA molecules. The miRNAdetection agents may be any suitable molecule that binds to and/or iscomplementary to the one or more miRNA molecules including, but notlimited to, complementary oligonucleotides, antisense oligonucleotides,and aptamers. The set of reagents maybe used in accordance with anymethod of measurement or quantification, such as those described above,and/or may be provided in a kit for detecting the set of one or moremiRNA molecules. According to some embodiments, the kit may include, inaddition to the set of reagents which contain miRNA detection agents, atleast one detection label to produce a detectable or visible signal(e.g., fluorescent labels, dyes, etc.), instructional materials, one ormore reference standards, additional agents (e.g., buffers,stabilizers), vessels for storing or transporting the detection agents,or a combination thereof.

According to some embodiments, the test level is compared to a referencelevel (or a “reference standard”) or a control level and the subject maythen be diagnosed as having cancer when the test level is significantlydifferent than the reference level or control level. A reference orcontrol level of an miRNA molecule may be any amount or a range ofamounts to be compared against the test level. For example, a referenceor control level of an miRNA molecule may be the level detected in apopulation of patients with a specified condition or disease (e.g.,malignancy, cancer or non-cancerous lung disease or condition) or thelevel detected in a control population of individuals without thecondition or disease. A control amount can be either in absolute amount(e.g., nanogram/mL or microgram/mL) or a relative amount (e.g., relativeintensity of signals).

In one embodiment, a test level of an miRNA molecule is considered to besignificantly different than a reference or control level if said testlevel is at least 1.5-fold higher (i.e., upregulated) or lower (i.e.,downregulated) as compared to a reference or control level of the miRNA.In some embodiments, the test level is considered to be significantlydifferent than the reference or control level is the test level is atleast 5-fold higher (i.e., upregulated) or lower (i.e., downregulated)as compared to the reference or control level. Alternatively, anincrease or decrease in an mRNA molecule is typically significantlydifferent if said increase or decrease has a p value of less than 0.5,or less than 0.05 (p<0.5 or p<0.05).

The one or more miRNA molecules described above may be part of an miRNAexpression signature or profile that is specific to a cancer stem cell,such as those discussed above. Such an expression signature may be usedin accordance with the methods herein to diagnose cancer, or may serveas a target for developing treatments for cancer. In one embodiment, themiRNA expression signature is specific to glioblastoma and includes, butis not limited to, one or more miRNA molecules selected from miR-10a,miR-10b, miR-140-3p, miR-140-5p, miR-204, miR-424, miR-34a, miR-193a-3p,miR-455-5p, miR-455-3p, miR-9, miR-10a, miR-148a, miR-488, miR-196a1,miR-182, miR-96, miR-193b, miR-27a, miR-196b, miR-10b, miR-29b2,miR-23a, miR-107, miR-542-3p, miR-93, miR-365a4, miR-450a, miR-100,miR-105, miR-363, miR-105, miR-106b, miR-15b, miR-21, miR-376c, miR-93,miR-99b, miR-155, miR-33a, miR-876-3p, miR-362-3p, miR-25, let-7i,miR-423-3p, miR-34b, miR-16-2, -miR-29a, miR-30d, miR-320, miR-181c,miR-128a, miR-21, let-7d, miR-450b-5p, miR-371-5p, miR-1245, miR-335,miR-492, miR-874, miR-30b, miR-193a-5p, miR-602, miR-346, miR-663,miR-25, miR-219-5p6, miR-184, miR-135a7, miR-584, miR-665, miR-638,miR-503, miR-628-3p, miR-381, miR-78, miR-92b, miR-149, miR-135b,miR-302d, miR-498, miR-766, miR-1389, miR-623, miR-519c-5p, miR-182,miR-494, miR-129-5p10, miR-513-5p, miR-200b, miR-634, miR-654-5p,miR-518b, miR-658, miR-373, miR-30c-2, miR-130a, miR-557, miR-551a,miR-637, miR-518c, miR-525-5p, miR-596, miR-552, miR-625, miR-183,miR-187, miR-544, miR-891a, miR-519e, miR-933, miR-939, miR-214,miR-671-5p, miR-137, miR-92b, miR-525-3p, miR-19a, and miR-409-5p.

In another embodiment, the miRNA expression signature includes one ormore miRNA molecules that are significantly upregulated in cancer stemcells (e.g., glioblastoma stem cells) and/or other cancer cells ascompared to healthy cells or healthy stem cells. The one or more miRNAmolecules that are significantly upregulated include, but are notlimited to, miR-10a, miR-10b, miR-140-3p, miR-140-5p, miR-204, miR-424,miR-34a, miR-193a-3p, miR-455-5p, miR-455-3p, miR-9, miR-10a, miR-148a,miR-488, miR-196a1, miR-182, miR-96, miR-193b, miR-27a, miR-196b,miR-10b, miR-29b2, miR-23a, miR-107, miR-542-3p, miR-93, miR-365a4,miR-450a, miR-100, miR-105, miR-363, miR-105, miR-106b, miR-15b, miR-21,miR-376c, miR-93, miR-99b, miR-155, miR-33a, miR-876-3p, miR-362-3p,miR-25, let-7i, miR-423-3p, miR-34b, miR-16-2, -miR-29a, miR-30d,miR-320, miR-181c, miR-128a, miR-21, let-7d, and miR-450b-5p. In oneembodiment, the one or more miRNA molecules that are significantlyupregulated include, but are not limited to miR-10a, miR-10b,miR-140-3p, miR-140-5p, miR-204, miR-424, miR-455-5p, miR-455-3p.

In another embodiment, the miRNA expression signature includes one ormore miRNA molecules that are significantly downregulated in cancer stemcells (e.g., glioblastoma stem cells) and/or other cancer cells ascompared to healthy cells or healthy stem cells. The one or more miRNAmolecules that are significantly downregulated include, but are notlimited to, miR-371-5p, miR-1245, miR-335, miR-492, miR-874, miR-30b,miR-193a-5p, miR-602, miR-346, miR-663, miR-25, miR-219-5p6, miR-184,miR-135a7, miR-584, miR-665, miR-638, miR-503, miR-628-3p, miR-381,miR-78, miR-92b, miR-149, miR-135b, miR-302d, miR-498, miR-766,miR-1389, miR-623, miR-519c-5p, miR-182, miR-494, miR-129-5p10,miR-513-5p, miR-200b, miR-634, miR-654-5p, miR-518b, miR-658, miR-373,miR-30c-2, miR-130a, miR-557, miR-551a, miR-637, miR-518c, miR-525-5p,miR-596, miR-552, miR-625, miR-183, miR-187, miR-544, miR-891a,miR-519e, miR-933, miR-939, miR-214, miR-671-5p, miR-137, miR-92b,miR-525-3p, miR-19a, and miR-409-5p. In one embodiment, the one or moremiRNA molecules that are significantly downregulated include, but arenot limited to hsa-miR-371-5p, hsa-miR-124-1, hsa-miR-124-2,hsa-miR-124-3, hsa-miR-335, hsa-miR-492, hsa-miR-874, hsa-miR-30b, andhsa-miR-602.

In certain embodiments, the miRNA expression signature includes at leastone miRNA molecule that is significantly upregulated and at least onemiRNA molecule that is significantly downregulated in cancer stem cells(e.g., glioblastoma stem cells) and/or other cancer cells as compared tohealthy cells or healthy stem cells. miRNA molecules that aresignificantly upregulated include, but are not limited to miR-10a,miR-10b, miR-140-3p, miR-140-5p, miR-204, miR-424, miR-455-5p,miR-455-3p; and miRNA molecules that are significantly downregulatedinclude, but are not limited to hsa-miR-371-5p, hsa-miR-124-1,hsa-miR-124-2, hsa-miR-124-3, hsa-miR-335, hsa-miR-492, hsa-miR-874,hsa-miR-30b, and hsa-miR-602.

The following examples are intended to illustrate various embodiments ofthe invention. As such, the specific embodiments discussed are not to beconstrued as limitations on the scope of the invention. It will beapparent to one skilled in the art that various equivalents, changes,and modifications may be made without departing from the scope ofinvention, and it is understood that such equivalent embodiments are tobe included herein. Further, all references cited in the disclosure arehereby incorporated by reference in their entirety, as if fully setforth herein.

EXAMPLES Example 1 Identification of a Set of miRNAs that areDifferentially Expressed in Glioblastoma Stem Cells and Normal NeuralStem Cells

The Examples described below show the results of a genome-wide miRNAexpression profiling study in human glioblastoma stem cells and normalneural stem cells using combined miRNA microarray and deep sequencinganalyses. This study led to the identification of eight miRNAs that aresubstantially up-regulated and two miRNAs that are significantlydown-regulated in glioblastoma stem cells, relative to normal neuralstem cells. Differential expression of four of these miRNAs, 2up-regulated and 2 down-regulated, was further validated by real-timeRT-PCR in both glioblastoma stem cells and glioblastoma patient tumortissues. Moreover, it was demonstrated that these up-regulated ordown-regulated miRNAs inhibit the expression of genes that are involvedin tumor suppression or tumorigenesis, respectively.

Materials and Methods

Ethics Statement.

The derivation of PBT003 (GSC1) and PBT017 (GSC2) has been described byBrown et al [Brown et al. 2009]. PBT707 (GSC3) is a de novo cell linederived from anonymized leftover tissues with the approval of the Cityof Hope Institutional Review Board. The study involves the use ofcompletely anonymized specimens. No informed consent is involved.NOD-scid IL2Rgamma^(null) (NSG) mice (6-8 weeks) were used forglioblastoma stem cell transplantation. Tumor cell transplantation wasperformed under an IACUC protocol approved by the City of HopeInstitutional Animal Care and Use Committee.

Glioblastoma Stem Cell and Neural Stem Cell Culture.

Glioblastoma stem cells were derived from newly diagnosed WHO grade IVglioblastoma tissues. Specifically, freshly isolated glioblastomatissues were minced with sterile scissors and dissociated into singlecells using 400 units/ml of collagenase III in DMEM/F12 mediumsupplemented with 5 μg/ml heparin, 1× B27 (GIBCO/BRL), and 2 mML-glutamine. Dissociated cells were then centrifuged at 1,200 rpm for 5min and the supernatant was discarded. To eliminate red blood cells, theresultant cells were incubated in 10 ml red blood cell lysis buffer(Invitrogen) for 10 min. Cells were centrifuged again at 1,200 rpm for 5min and supernatant was discarded. The resultant cells were resuspendedin DMEM/F12 medium supplemented with 20 ng/ml EGF, 20 ng/ml FGF, 5 μg/mlheparin, 1× B27 (GIBCO/BRL), and 2 mM L-glutamine and cultured in thismedium thereafter. Tumor spheres appeared around one week in culture.Normal human neural stem cells were derived from primary human braintissues and maintained in the same culture media. Specifically, humanfetal brain tissues (Biosciences Resources) were dissociated in coldHanks balance salt solution (HBSS) using polished glass pipette. Theresultant cells were centrifuged and resuspended in DMEM/F12 mediumsupplemented with 0.5× B27, 25 μg/ml insulin, 20 μg/ml apo-transferrin,30 nM sodium selenite, 20 nM progesterone, 100 mM putrescine, 20 ng/mlFGF and 10 ng/ml LIF. The initial culture was split at 1:2 each day for4 days, followed by media change every other day till day 21. Humanneurospheres started to appear around day 14. The spheres were splitaround day 21 with Accutase (Sigma) and cultured in DMEM/F12 mediumsupplemented with 20 ng/ml EGF, 20 ng/ml FGF, 5 μg/ml heparin, 1× B27(GIBCO/BRL), and 2 mM L-glutamine thereafter. Both tumor spheres andnormal neurospheres were characterized for their self-renewal andmultipotency. Glioblastoma stem cell spheres were also characterized fortheir ability to derive brain tumors.

For differentiation, both glioblastoma stem cells and neural stem cellswere induced into differentiation using 0.5% fetal bovine serum and 1 μMall-trans retinoic acid. For in vivo tumor formation assays, 2×10⁵dissociated glioblastoma stem cells were injected into cerebral cortexof NSG mice by stereotaxic injection. The coordinates for the injectionwere AP 0.6 mm, ML+1.6 mm and DV −2.6 mm. Brains were harvested 5 weeksafter cell transplantation. Frozen brains were cut into 20 μm coronalsections, followed by Hematoxylin & Eosin (H&E) staining.

Glioblastoma Stem Cell Transfection Using a Dendrimer-Based DeliverySystem.

Spheres of glioblastoma stem cells were dissociated and seeded into24-well plates at 2×10⁵ cells per well in 300 μl of medium. Thegeneration-5 (G5) dendrimers and Opti-MEM solution were mixed by vortexfor 10 seconds, and incubated at room temperature (RT) for 10 min. ThemiRNA duplexes or the combination of miRNAs and their short hairpin RNAinhibitors were added into dendrimer/Opti-MEM solution in a total volumeof 100p1, mixed gently for 10 sec, and incubated at RT for 25 min. Thenitrogen-to-phosphorus (N/P) ratio of the dendrimer/RNA complex is 5.The 100 μl dendrimer/RNA complex was added into 300 μl cell suspensionin each well of 24-well plates, shake gently and put back to CO₂incubator. Forty-eight hr after transfection, cells were collected andsubjected to Western blot analysis.

Western Blot Analysis.

Whole cell extracts of glioblastoma stem cells were prepared using RIPAbuffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP40, 0.5% deoxycholateand 0.1% SDS) containing protease inhibitor cocktail (Roche). Westernblotting was performed with anti-NRAS (sc-31, 1:100) and anti-PIM3(sc-98959, 1:100) antibodies from Santa Cruz.

Glioblastoma Stem Cell Transplantation.

NSG mice (6-8 weeks) were used for glioblastoma stem celltransplantation. Tumor cell transplantation was performed under theIACUC protocol 05050 approved by the City of Hope Institutional AnimalCare and Use Committee. 5×10⁴ dissociated glioblastoma stem cells wereinjected into the front lobe of forebrains by stereotaxic injection. Thecoordinates for the injection were AP 0.6 mm, ML+1.6 mm and DV −2.6 mm.

Reporter Construct Preparation.

DNA fragments encoding the 3′ UTR of putative miRNA targets were clonedinto psiCHECK 2 (Promega), downstream of a Renilla luciferase reportergene. The PCR primers that were used for 3′ UTR cloning of each gene areas follows: CSMD1 forward: 5′ GAT CCT CGA GCT GTT CTG TCG CAG AAT G 3′(SEQ ID NO: 13) and CSMD1 reverse: 5′ GAT CGC GGC CGC GTC AGC ATT TTGCAC CTA G3′ (SEQ ID NO: 14); PIM3 forward: 5′ GAT CCT CGA GGC TTG TGAGGA GCT GCA C 3′ (SEQ ID NO: 15) and PIM3 reverse: 5′ GAT CGC GGC CGCGGA AAC TTG TCA GGT CAC C 3′ (SEQ ID NO: 16); NRAS forward: 5′ GAT CCTCGA GCT GGA GGA GAA GTA TTC CTG 3′ (SEQ ID NO: 17) and NRAS reverse: 5′GAT CGC GGC CGC TGC AAA TGT AGA GCT TTC TGG 3′ (SEQ ID NO: 18).Corresponding miRNA binding sites on the 3′ UTRs were mutated bysite-directed mutagenesis according to the manufacturer's instructions(Stratagene). The binding site of hsa-miR-124 on the 3′ UTR of PIM3 andNRAS was mutated from GTGCCTT to GTGGACA; the binding site ofhsa-miR-10b on the 3′ UTR of CSMD1 was mutated from ACAGGGT to ACAGTCC.

Transfection and Reporter Assay.

Plasmid DNA or DNA-miRNA mixture was transfected into HEK293 cells usingTransfectin (Bio-Rad) as described [Zhao et al. 2009; Zhao et al. 2010;Sun et al. 2011]. miR-10a, miR-10b or miR-124 RNA duplexes and/or theircorrespondent RNA inhibitors (Dharmacon) were mixed in 50 μl serum freemedia with Transfectin, incubated at RT for 20 min. Negative controlsfor miRNA and their hairpin inhibitors were included. The finalconcentration of miRNAs or their inhibitors was 20 nM. The resultantmixture was added dropwise to HEK293 cells in a 24-well plate with 450μl medium per well to a total volume of 500 μl per well. The transfectedcells were harvested 48 h after transfection and subjected to subsequentreporter assays as described [Sun et al. 2007]. Reporter Renillaluciferase activity was measured 48 hrs after transfection using DualLuciferase Assay kit (Promega). The Renilla luciferase activity wasnormalized by firefly luciferase internal control and expressed asrelative luciferase activity. The miR-10a RNA duplex sense sequence is5′ TAC CCT GTA GAT CCG AAT TTG TG 3′ (SEQ ID NO: 19). The miR-10b RNAduplex sense sequence is 5′ TAC CCT GTA GAA CCG AAT TTG TG 3′ (SEQ IDNO: 20). The miR-124 RNA duplex sense sequence is 5′ TAA GGC ACG CGG TGAATG CC 3′ (SEQ ID NO: 21). And the control RNA duplex sense sequence is5′ UCA CAA CCU CCU AGA AAG AGU AGA 3′ (SEQ ID NO: 22).

Real-Time RT-PCR Analysis.

For miRNA expression, total RNAs were reversely transcribed andquantified by real-time RT-PCR with TaqMan MicroRNA Assay kit (AppliedBiosystems). The expression of specific miRNAs was normalized usinghuman U18 snRNA. For mRNA expression, putative miRNA targets werequantified by iTaq SYBR Green Supermix with ROX (Bio-Rad). Primers usedfor RT-PCR include PIM3 forward: 5′ AGC TCA AGC TCA TCG ACT TC 3′ (SEQID NO: 23) and PIM3 reverse: 5′ TAG CGG TGG TAG CGG ATC 3′ (SEQ IDNO:24); NRAS forward: 5′ CCA TGA GAG ACC AAT ACA TGA G 3′ (SEQ ID NO:25) and NRAS reverse: 5′ GCT TAA TCT GCT CCC TGT AG 3′ (SEQ ID NO: 26);HOXD10 forward: 5′ TTC CCG AAG AGA GGA GCT G 3′ (SEQ ID NO: 27) andHOXD10 reverse: 5′ CTG CCA CTC TTT GCA GTG AG 3′ (SEQ ID NO: 28); CSMD1forward: 5′ GCA GAA ATG CTT ACT GAG GAT G 3′ (SEQ ID NO: 29) and CSMD1reverse: 5′ AGA ACC CTC AAA CTG CAA CTG 3′ (SEQ ID NO: 30); GAPDHforward: 5′ ATC ACC ATC TTC CAG GAG C 3′ (SEQ ID NO: 31) and GAPDHreverse 5′ CCT TCT CCA TGG TGG TGA AG 3′ (SEQ ID NO: 32).

miRNA Microarray and Deep Sequencing Analysis.

Total RNAs were extracted from glioblastoma stem cells or human neuralstem cells by TRIzol (Invitrogen) method according to manufacturer'sprotocol. Ten μg of RNA was used for miRNA microarray using Exiqonplatform. One μg of RNA was used for deep sequencing using IlluminaGenome Analyser II (GAII). All data are MIAME compliant.

Pathway Analysis.

Common putative targets of either the down-regulated or the up-regulatedmiRNAs were uploaded onto the Database for Annotation, Visualization andIntegrated Discovery (DAVID) Functional Annotation BioinformaticsMicroarray Analysis (http://david.abcc.ncifcrf.gov/). Kyoto Encyclopediaof Genes and Genomes (KEGG) pathway in DAVID was used to depict thebiological meanings of the common miRNA targets.

Results

Differential miRNA Expression in Glioblastoma Stem Cells and NormalNeural Stem Cells.

To identify miRNAs that are differentially expressed in glioblastomastem cells and normal neural stem cells, three primary glioblastoma stemcell lines and three normal human neural stem cell lines wereestablished to determine if miRNA expression was significantly differentin tumor stem cells as compared to normal stem cells. Human primaryglioblastoma stem cells were derived from newly diagnosed glioblastomamultiforme IV patients and cultured in DMEM/F12 media supplemented withepithelial growth factor (EGF), fibroblast growth factor (FGF), and B27supplement. Human normal neural stem cells were derived from normalhuman brain tissues and cultured in the same media. Both glioblastomastem cells and normal neural stem cells grew as neurospheres under theculture conditions (FIG. 1A). Both types of cells are multipotent,having the ability to differentiate into Tuj1-positive neurons and/orGFAP-positive astrocytes when induced into differentiation using fetalbovine serum and all-trans retinoic acid (FIG. 1B). However, theglioblastoma stem cells were able to generate tumors when transplantedto the immunodeficient NSG mice (FIG. 1C), whereas the human neural stemcells did not (data not shown).

Combined microarray and deep sequencing analyses were performed todetermine the expression profile of miRNAs in glioblastoma stem cellsand normal neural stem cells. Total RNAs were prepared from bothglioblastoma stem cells and neural stem cells for miRNA microarrayanalysis. In microarray analysis, 10 miRNAs were identified as havingmore than a 5-fold up-regulation in expression in glioblastoma stemcells and 8 miRNAs were identified as having more than a 5-folddown-regulation in expression in glioblastoma stem cells, relative toneural stem cells (Table 1). The differentially expressed miRNAs thatexhibit more than 1.5-fold difference in the expression betweenglioblastoma stem cells and neural stem cells are shown in Table 2below.

TABLE 1 Up-regulated and down-regulated miRNAs in human glioblastomastem cells, compared to human neural stem cells. Chromosomal miRNAlocation Fold-Change p-value up-regulated hsa-miR-10a 17q21.32 93.653.28E−10 hsa-miR-10b 2q31.1 90.38 2.06E−09 hsa-miR-140-3p 16q22.1 14.101.62E−10 hsa-miR-140-5p 16q22.1 12.19 5.56E−09 hsa-miR-204 9q21.12 9.054.08E−08 hsa-miR-424 Xq26.3 8.38 2.53E−08 hsa-miR-455-5p 9q32 5.871.48E−05 hsa-miR-455-3p 9q32 5.41 9.32E−05 down-regulated hsa-miR-371-5p19q13.42 −15.27 8.52E−11 hsa-miR-124-1* 8p23.1 hsa-miR-124-2* 8q12.3−13.37 8.33E−05 hsa-miR-124-3* 20q13.33 hsa-miR-335 7q32.2 −13.241.43E−09 hsa-miR-492 12q22 −7.77 5.38E−08 hsa-miR-874 5q31.2 −6.761.53E−08 hsa-miR-30b* 8q24.22 −6.54 2.73E−09 hsa-miR-602 9q34.3 −5.752.63E−08 *hsa-miR-124 is transcribed from three chromosomal locations,but the mature sequences are the same.

TABLE 2 Up-regulated and down-regulated miRNAs (>1.5 fold) in humanglioblastoma stem cells, compared to human neural stem cells.Chromosomal miRNA location Fold-Change p-value Up-regulated hsa-miR-10a17q21.32 93.64622 3.28E−10 hsa-miR-10b 2q31.1 90.38293 2.06E−09hsa-miR-140-3p 16q22.1 14.10262 1.62E−10 hsa-miR-140-5p 16q22.1 12.193255.56E−09 hsa-miR-204 9q21.12 9.05353 4.08E−08 hsa-miR-424 Xq26.3 8.383822.53E−08 hsa-miR-34a 1p36.22 7.73283 2.21E−07 hsa-miR-193a-3p 17q11.26.39914 7.95E−06 hsa-miR-455-5p 9q32 5.87119 1.48E−05 hsa-miR-455-3p9q32 5.40680 9.32E−05 hsa-miR-9* 1q22 4.43204 8.28E−08 hsa-miR-10a*17q21.32 3.89600 1.92E−05 hsa-miR-148a 7p15.2 3.16202 1.93E−05hsa-miR-488 1q25.2 2.77759 1.69E−03 hsa-miR-196a¹ 17q21.32 2.765212.69E−03 hsa-miR-182 7q32.2 2.75689 1.52E−03 hsa-miR-96 7q32.2 2.614001.61E−03 hsa-miR-193b 16p13.12 2.57981 4.90E−06 hsa-miR-27a 19p13.132.53543 1.73E−07 hsa-miR-196b 7p15.2 2.50868 3.54E−03 hsa-miR-10b*2q31.1 2.40614 8.55E−03 hsa-miR-29b² 7q32.3 2.39299 3.27E−07 hsa-miR-23a19p13.13 2.34219 9.88E−08 hsa-miR-107 10q23.31 2.31374 4.42E−07hsa-miR-542-3p Xq26.3 2.28873 4.59E−03 hsa-miR-9³ 1q22 2.24593 1.27E−07hsa-miR-365a⁴ 16p13.12 2.20050 2.54E−06 hsa-miR-450a Xq26.3 2.110761.43E−02 hsa-miR-100 11q24.1 2.04624 5.71E−07 hsa-miR-105 Xq28 1.995293.86E−03 hsa-miR-363 Xq26.2 1.99346 1.23E−02 hsa-miR-105* 4q24 1.881561.57E−02 hsa-miR-106b 7q22.1 1.86896 5.63E−06 hsa-miR-15b 3q25.331.79677 3.00E−06 hsa-miR-21 17q23.1 1.76837 3.03E−05 hsa-miR-376c14q32.31 1.76028 5.72E−04 hsa-miR-93 7q22.1 1.74398 6.15E−06 hsa-miR-99b19q13.41 1.73839 1.77E−05 hsa-miR-155 21q21.3 1.72212 1.59E−02hsa-miR-33a 22q13.2 1.72056 8.82E−05 hsa-miR-876-3p 9p21.1 1.686694.45E−02 hsa-miR-362-3p Xp11.23 1.67653 4.12E−02 hsa-miR-25 7q22.11.66555 1.31E−04 hsa-let-7i 12q14.1 1.66413 1.12E−05 hsa-miR-423-3p17q11.2 1.64838 2.50E−04 hsa-miR-34b 11q23.1 1.62786 9.72E−05hsa-miR-16-2* 3q25.33 1.62586 1.81E−03 hsa-miR-29a 7q32.3 1.615839.27E−06 hsa-miR-30d 8q24.2 1.61189 9.38E−04 hsa-miR-320 8p21.3 1.608648.71E−05 hsa-miR-181c 19p13.13 1.56262 1.30E−02 hsa-miR-128a 2q21.31.55521 4.20E−02 hsa-miR-21* 17q23.1 1.54998 2.94E−02 hsa-let-7d 9q22.321.53430 1.10E−03 hsa-miR-450b-5p Xq26.3 1.53397 3.49E−02 Down-regulatedhsa-miR-371-5p 19q13.42 −15.26715 8.52E−11 hsa-miR-124⁵ 8p23.1 −13.372368.33E−05 hsa-miR-335 7q32.2 −13.23553 1.43E−09 hsa-miR-492 12q22−7.76626 5.38E−08 hsa-miR-874 5q31.2 −6.76244 1.53E−08 hsa-miR-30b*8q24.22 −6.54116 2.73E−09 hsa-miR-193a-5p 17q11.2 −5.76493 2.38E−08hsa-miR-602 9q34.3 −5.74746 2.63E−08 hsa-miR-346 10q23.2 −5.697124.97E−02 hsa-miR-663 20p11.1 −5.37200 1.31E−02 hsa-miR-25* 7q22.1−4.97894 2.47E−06 hsa-miR-219-5p⁶ 6p21.32 −4.91797 5.92E−07 hsa-miR-18415q25.1 −4.87568 4.42E−08 hsa-miR-135a⁷ 3p21.1 −4.87064 1.94E−07hsa-miR-584 5q32 −4.60287 1.77E−08 hsa-miR-665 14q32.2 −4.35369 7.57E−09hsa-miR-638 19p13.2 −3.47384 3.88E−04 hsa-miR-503 Xq26.3 −3.435021.20E−08 hsa-miR-628-3p 15q21.3 −3.42905 1.71E−07 hsa-miR-381 14q32.31−3.33814 2.55E−07 hsa-miR-7⁸ 9q21.32 −2.91031 4.96E−04 hsa-miR-92b 1q22−2.90543 2.73E−07 hsa-miR-149* 2q37.3 −2.87318 1.44E−03 hsa-miR-135b1q32.1 −2.87250 4.47E−07 hsa-miR-302d* 4q25 −2.77368 2.82E−03hsa-miR-498 19q13.42 −2.75015 2.70E−03 hsa-miR-766 Xq24 −2.499791.89E−03 hsa-miR-138⁹ 3p21.32 −2.48259 9.79E−07 hsa-miR-623 13q32.3−2.43864 3.92E−03 hsa-miR-519c-5p 19q13.42 −2.38133 1.61E−05hsa-miR-182* 7q32.2 −2.26786 4.18E−02 hsa-miR-494 14q32.31 −2.154543.51E−06 hsa-miR-129-5p¹⁰ 7q32.1 −2.13428 6.47E−04 hsa-miR-513-5p11q23.1 −2.12005 9.55E−03 hsa-miR-200b* 1p36.33 −2.04703 5.69E−05hsa-miR-634 17q24.2 −2.02966 6.14E−06 hsa-miR-654-5p 14q32.31 −2.016637.48E−04 hsa-miR-518b 19q13.42 −1.98208 1.05E−03 hsa-miR-658 22q13.1−1.94699 1.80E−06 hsa-miR-373* 19q13.42 −1.91189 4.25E−03 hsa-miR-30c-2*6q13 −1.88988 2.12E−06 hsa-miR-130a 11q12.1 −1.87039 3.78E−05hsa-miR-557 1q24.2 −1.83994 1.62E−03 hsa-miR-551a 1p36.32 −1.821152.53E−03 hsa-miR-637 19p13.3 −1.81083 2.07E−02 hsa-miR-518c* 19q13.42−1.77801 4.15E−05 hsa-miR-525-5p 19q13.42 −1.75943 4.76E−02 hsa-miR-5968p23.3 −1.74891 1.75E−03 hsa-miR-552 1p34.3 −1.72726 7.31E−04hsa-miR-625* 14q23.3 −1.71695 7.00E−04 hsa-miR-183* 7q32.2 −1.707811.41E−03 hsa-miR-187* 18q12.2 −1.70468 1.12E−02 hsa-miR-544 14 −1.692684.74E−02 hsa-miR-891a Xq27.3 −1.67598 1.29E−02 hsa-miR-519e* 19q13.42−1.67235 1.56E−02 hsa-miR-933 2q31.1 −1.66767 5.19E−05 hsa-miR-9398q24.3 −1.66214 5.40E−03 hsa-miR-214 1q24.3 −1.64500 6.92E−03hsa-miR-671-5p 7q36.1 −1.64192 7.94E−05 hsa-miR-137 1p21.3 −1.630143.96E−02 hsa-miR-92b* 1q22 −1.54966 4.57E−02 hsa-miR-525-3p 19q13.42−1.54729 1.45E−02 hsa-miR-19a 13q31.3 −1.51239 1.90E−04 hsa-miR-409-5p14q32.31 −1.51024 5.12E−03 ¹⁻¹⁰For miRNAs that have more than oneprimary precursors, the chromosomal location of the first primaryprecursor is shown in the table. The chromosomal locations of otherprimary miRNAs include: 1) hsa-miR-196a-2: 12q13.13; 2) hsa-miR-29b-2:1q32.2; 3) hsa-miR-9-2: 5q14.3, hsa-miR-9-3: 15q26.1; 4) hsa-miR-365b:17q11.2; 5) hsa-miR-124-2: 8q12.3, hsa-miR-124-3: 20q13.33; 6)hsa-miR-219-2-5p: 9q34.11; 7) hsa-miR-135a-2: 12q23.1; 8) hsa-miR-7-2:15q26.1, hsa-miR-7-3: 19p13.3; 9) hsa-miR-138-2: 16q13; and 10)hsa-miR-129-2-5p: 11p11.2.

Using an Illumina Genome Analyzer II (GAII) sequencing system,whole-genome small RNA sequencing was performed in glioblastoma stemcells and neural stem cells. Significantly more miRNAs were detected tobe differentially expressed in glioblastoma stem cells and neural stemcells in deep sequencing analysis. For example, deep sequencing analysisrevealed 105 miRNAs that were up-regulated more than 5-fold inglioblastoma stem cells. However, microarray analysis revealed only 10miRNAs showing more than 5-fold increase in glioblastoma stem cells.Interestingly, 8 out of the 10 miRNAs that were up-regulated more than5-fold in microarray analysis also exhibited significantly increasedexpression in deep sequencing analysis (Table 3). Two of the miRNAs thathad more than 5-fold decrease of expression in microarray analysis alsoshowed more than 5-fold reduction of expression in deep-sequencinganalysis (Table 3). Taken together, the combined results of themicroarray and deep sequencing analyses identified a set of miRNAs thatare differentially expressed in glioblastoma stem cells as compared tonormal neural stem cells.

TABLE 3 The miRNA signature of glioblastoma stem cells identified usingboth microarray and deep sequencing analyses. Microarray Deep sequencingFold change p value Fold change p value Up-regulated hsa-miR-10a 93.65  <1E−07 35,949 0.00E+00 hsa-miR-10b 90.38   <1E−07 4,128 0.00E+00hsa-miR-140-5p 12.19   <1E−07 7.2 0.00E+00 hsa-miR-204 9.05   <1E−07 50.00E+00 hsa-miR-424 8.38   <1E−07 66 0.00E+00 hsa-miR-34a 7.73 2.00E−072.5 4.00E−240 hsa-miR-193a-3p 6.4 8.00E−06 93 9.00E−21 hsa-miR-455-5p5.87 1.00E−05 5.3 5.00E−214 Down-regulated hsa-miR-124 −13.37 8.00E−05−10 5.00E−80 hsa-miR-874 −6.76   <1E−07 −33 1.50E−98

Validation of the Differentially Expressed miRNAs Using Real-TimeRT-PCR. The distinct expression of these miRNAs in glioblastoma stemcells and neural stem cells was further validated using real-time RT-PCRanalysis. RT-PCR results of the top three miRNAs that are up-regulatedin glioblastoma stem cells in both microarray and deep sequencinganalyses (Table 3) are shown in FIG. 2A-C. All three miRNAs showed asignificant up-regulation in the three primary glioblastoma stem celllines (GSC1-3) tested, compared to three lines of normal neural stemcells. miR-10a revealed a dramatic increase of expression in all threeglioblastoma stem cell lines tested, with more than 100-foldup-regulation of expression in two of the glioblastoma stem cell lines(FIG. 2A). miR-10b exhibited even higher expression in glioblastoma stemcell lines GSC1 and GSC3, with up to 2,505-fold increase of expressionin GSC1 (FIG. 2B). miR-140-5p also displayed significant increase ofexpression in all three glioblastoma stem cell lines tested, althoughwith much lower fold induction (FIG. 2C).

As discussed above, two miRNAs are down-regulated more than 5-fold inglioblastoma stem cells as shown by both microarray and deep sequencinganalyses. The expression of these two miRNAs was also validated usingreal-time RT-PCR assays. Both miR-124 and miR-874 exhibited reproducibledecrease of expression in all three glioblastoma stem cell lines tested,compared to normal neural stem cells (FIG. 2D, E). Specifically,miR-874, which has not been well characterized to date, exhibited asignificant reduction of expression in all three glioblastoma stem celllines, with more than 20-fold reduction in two of the glioblastoma stemcell lines GSC1 and GSC3 (FIG. 2E).

In addition, normal and glioblastoma brain tissues were analyzed todetermine whether the set of miRNAs identified above are differentiallyexpressed in distinct expression patterns as shown in the cultured cellstudies descried above. For this purpose, RNAs were isolated from 9grade IV glioblastoma multiforme brain tissue samples and 4 non-tumornormal brain tissue samples. Real-time RT-PCR analyses were performed todetect the expression of two up-regulated miRNAs and two down-regulatedmiRNAs. Consistent with the results from tumor stem cells describedabove, miR-10a exhibited a substantial increase of expression in mostglioblastoma tissues (FIG. 3A). miR-10b also exhibited a substantialup-regulation of expression in all of the glioblastoma tissues tested,with an average increase of 142-fold (FIG. 3B). For miRNAs that weredown-regulated in glioblastoma stem cells, both miR-124 and miR-874displayed a significant decrease of expression in most of theglioblastoma tissues tested, compared to their average expression innormal brain tissues (FIG. 3C, D).

Target Identification of the Differentially-Expressed miRNAs.

By using a Targetscan algorithm [Lewis et al. 2003], CUB and SUSHImultiple domain protein 1 (CSMD1) were identified as candidatedownstream targets for miR-10a and miR-10b, the most highly up-regulatedmiRNAs in glioblastoma stem cells in the profiling analyses. CSMD1 is atumor suppressor gene that maps to chromosome 8p23, a region deleted inmany tumor types [Kamal et al. 2010]. Sequence analysis revealed thatthe seed region of both miR-10a and miR-10b could form complementarybase pairs with the 3′ untranslated region (3′ UTR) of human and mouseCSMD1 mRNAs (FIG. 4A, B). To demonstrate a direct interaction betweenthe 3′ UTR of CSMD1 and miR-10 (miR10a and miR-10b), the 3′ UTR regionof human CSMD1 that contains the putative miR-10 recognition sites andflanking sequences was inserted downstream of a Renilla luciferasereporter gene into a siCheck vector. RNA duplexes of mature miR-10a ormiR-10b were transfected into human embryonic kidney HEK293 cells alongwith the reporter gene. Significant repression of the reporter gene wasobserved in both miR-10a and miR-10b-transfected cells (FIG. 4C, D).Mutation of the miR-10 targeting sites abolished the repression (FIG.4C, D). Furthermore, treatment of the inhibitors of miR-10a and miR-10breversed the inhibitory effect of miR-10a and miR-10b on the luciferasereporter activity, respectively (FIG. 4C, D). These results suggest thatboth miR-10a and miR-10b repress CSMD1 expression through the predictedtargeting sites in CSMD1 3′ UTR.

Since miR-10a and miR-10b are both up-regulated in glioblastoma stemcells relative to neural stem cells, the expression of CSMD1 wasexamined in both cell types. Dramatic reduction of CSMD1 mRNA expressionwas detected in glioblastoma stem cells by RT-PCR analysis, compared toneural stem cells (FIG. 4E), consistent with the observation that CSMD1expression is repressed by miR-10 (FIG. 4C, D). The homeoboxtranscription factor HOXD10 has been identified as a tumor suppressorgene targeted by miR-10b in breast cancers [Ma et al. 2007]. Here, itwas shown that the HOXD10 mRNA expression is also dramatically reduced(>20-fold) in glioblastoma stem cells examined, compared to normalneural stem cells (FIG. 4F). Together, these results suggest that miR-10targets the expression of tumor suppressor genes, CSMD1 and HOXD10, inglioblastoma stem cells.

Furthermore, using the Targetscan algorithm, the oncogenes NRAS and PIM3were selected as putative target genes of miR-124, one of thedown-regulated miRNAs in glioblastoma stem cells. NRAS is a smallguanine-nucleotide binding protein and one of the three RAS (KRAS, NRAS,HRAS) isoforms [Kiessling et al. 2011]. The RAS signaling pathway playsan important role in many cancers by regulating cell proliferation,differentiation, and survival [Kan et al. 2010]. Using Targetscanalgorithm, miR-124 was predicted to have a targeting site at the 3′ UTRof the NRAS gene. This targeting site is conserved in human, mouse, anddog NRAS (FIG. 5A). To validate the targeting of NRAS by miR-124, aluciferase reporter construct with human NRAS 3′ UTR containing thepredicted miR-124 targeting site was made, and the flanking sequencesinserted into the 3′UTR of a Renilla luciferase reporter gene in asiCHECK vector. Transfection of miR-124 RNA duplexes led to significantrepression of the reporter gene (FIG. 5B). Mutation of the putativemiR-124 targeting site abolished the repression (FIG. 5B). Furthermore,treatment with a miR-124 inhibitor reversed the inhibitory effect ofmiR-124 on the luciferase reporter activity (FIG. 5B). These resultssuggest that miR-124 represses NRAS expression through the predictedtargeting site in NRAS 3′UTR.

Next, it was tested whether miR-124 targets NRAS expression inglioblastoma stem cells. Mature miR-124 RNA duplexes were introducedinto GSC1 cells using a cationic triethanolamine-core polyamidoamine(PAMAM) dendrimer-mediated small RNA delivery system [Zhou et al. 2006;Zhou et al. 2011]. A control RNA duplex was included as a negativecontrol. NRAS expression levels were examined by Western blot analysis.Reduction of NRAS protein level was detected in miR-124-transfectedcells. Co-transfection of a miR-124 RNA inhibitor abolished theinhibitory effect of miR-124 on NRAS expression (FIG. 5C). This resultindicates that miR-124 down-regulates endogenous NRAS expression inglioblastoma stem cells.

The expression of NRAS in glioblastoma stem cells and neural stem cellswas examined, where miR-124 exhibits differential expression. Asignificant increase of NRAS mRNA expression was detected inglioblastoma stem cells, compared to neural stem cells (FIG. 5D). Theinverse expression pattern of NRAS and miR-124 is consistent with theobservation that NRAS expression is repressed by miR-124 (FIG. 5B, C).

A putative targeting site of miR-124 was also identified in the 3′ UTRof both human and mouse PIM3, a proto-oncogene with serine/threoninekinase activity (FIG. 5E). PIM3 has been shown to promote tumor cellgrowth through modulating cell cycle regulators [Wu et al. 2010; Braultet al. 2010]. To validate the targeting of PIM3 by miR-124, a luciferasereporter construct with human PIM3 3′ UTR was made, which contained thepredicted miR-124 targeting site and the flanking sequences insertedinto the 3′UTR of a Renilla luciferase reporter gene. Transfection ofmiR-124 led to significant repression of the reporter gene and mutationof the putative miR-124 targeting site abolished the repression (FIG.5F). Furthermore, treatment with a miR-124 inhibitor reversed theinhibitory effect of miR-124 on the luciferase reporter activity (FIG.5F). These results suggest that miR-124 represses PIM3 expressionthrough the predicted targeting site in its 3′ UTR.

To test whether miR-124 targets PIM3 expression in glioblastoma stemcells, mature miR-124 RNA duplexes were introduced into GSC1 cells usingthe dendrimer-mediated delivery system [Zhou et al. 2006; Zhou et al.2011]. A control RNA duplex was included as a negative control.Reduction of PIM3 protein level was detected in miR-124-transfectedcells by Western blot analysis. Co-transfection of a miR-124 RNAinhibitor abolished the inhibitory effect of miR-124 on PIM3 expression(FIG. 5G). This result indicates that miR-124 down-regulates endogenousPIM3 expression in glioblastoma stem cells. Moreover, a significantincrease of PIM3 mRNA expression was detected in the glioblastoma stemcells tested, compared to neural stem cells (FIG. 5H), furthersupporting the idea that miR-124 represses PIM3 expression.

It is increasingly clear that miRNAs are important regulators of keysignaling pathways implicated in tumorigenesis. Using Kyoto Encyclopediaof Genes and Genomes (KEGG) pathway analysis, the predicted targets ofmiRNAs that showed more than 5-fold up-regulation or down-regulation inglioblastoma stem cells in the microarray analysis were compared. Sevenof the ten miRNAs that were up-regulated more than 5-fold inglioblastoma stem cells are predicted to have components of the p53pathway as common targets (FIG. 6A). The p53 pathway has been shown tobe involved in cell cycle arrest, apoptosis, inhibition of cellmigration, inhibition of angiogenesis, and affect genomic stability[Junttila & Evan 2009]. In contrast, five out of eight miRNAs thatexhibited more than 5-fold down-regulation in glioblastoma stem cellswere predicted to target components of the IGF pathway (FIG. 6B) thathas been implicated in promoting cell growth, survival and migration[Clayton et al. 2011].

Discussion

The present study investigated genome-wide miRNA expression in tumorstem cell populations of glioblastoma, the most frequent and malignantprimary brain tumor. In spite of recent improvement of surgical andradiotherapeutic techniques, the prognosis for glioblastoma patients isstill very poor. The search for molecular targets is fundamental todevelop effective treatments for glioblastoma.

Global profiling is an effective approach to identify abnormallyexpressed miRNAs in tumor genomes. Three different technical platformswere used to determine the differential expression of miRNAs inglioblastoma stem cells and neural stem cells. The microarray platformwas combined with the newly emerged small RNA deep sequencing technologyto profile miRNA expression in glioblastoma stem cells and normal neuralstem cells and validated the profiling results using quantitativeRT-PCR. Although the absolute fold change obtained from each platform isdifferent due to the different sensitivity of the techniques, the trendof the change for the miRNAs studied is consistent. The miRNA expressionprofile could clearly distinguish glioblastoma stem cells from normalneural stem cells, allowing us to identify a miRNA signature ofglioblastoma stem cells that were significantly up-regulated ordown-regulated in glioblastoma stem cells, relative to neural stemcells.

In line with the findings that a set of miRNAs are differentiallyexpressed in glioblastoma stem cells and normal neural stem cells,certain miRNAs also exhibit distinct expression profiles in glioblastomatissues and normal brain tissues. For example, it was demonstrated thatthe expression of miR-874 is dramatically reduced in glioblastomatissues, compared to normal brain tissues. miR-124, another miRNA thatwas down-regulated in glioblastoma stem cells, also exhibited reducedexpression in glioblastoma tissues in this study, consistent with theresults of previous glioblastoma tumor tissue profiling [Huse et al.2009; Silber et al. 2008; Ciafre et al. 2005; Godlewski et al. 2008; Xiaet al. 2012; Skalsky & Cullen 2011; Fowler et al. 2011].

In this study, miR-10b was shown to be highly expressed in bothglioblastoma stem cells and in glioblastoma tumor tissues. Up-regulationof miR-10b was also observed in other glioblastoma samples [Huse et al.2009; Ciafre et al. 2005; Godlewski et al. 2008], suggesting animportant role for miR-10b in glioblastoma tumorigenesis. Moreover, arecent study revealed that miR-10b expression is inversely correlatedwith glioblastoma patient survival [Gabriely et al. 2011].Interestingly, miR-10b was also found to be up-regulated in breastcancer, leukemia, and pancreatic cancer and promote tumor invasion andmetastasis in breast cancer [Ma et al. 2007; Calin et al. 2004;Bloomston et al. 2007]. Together, these results suggest that somemiRNAs, such as miR-10b, may function as a global oncogene to stimulatetumorigenesis in multiple tissues. Likewise, miR-124 is also frequentlydown-regulated in other cancers, such as medulloblastoma, hepatocellularcarcinoma, and oral squamous carcinoma [Li et al. 2009; Furuta et al.2010; Hunt et al. 2011], suggesting that it may function as a generaltumor suppressor. Therefore the knowledge of miRNAs that we haveobtained for glioblastoma stem cells may be applicable to other types ofcancer stem cells.

Pathway analysis revealed that most of the significantly up-regulatedmiRNAs, with more than 5-fold increase in glioblastoma stem cells asshown in the microarray analysis described herein, have putative targetsin a common pathway, the p53 pathway. The dysregulation of the p53pathway has been shown to be an underlying mechanism for tumorigenesis[Junttila & Evan 2009], thus the up-regulated miRNAs may function asoncomiRs by targeting the p53 pathway if their role in regulating thep53 pathway is confirmed. On the other hand, most of the down-regulatedmiRNAs, with more than 5-fold decrease in glioblastoma stem cells, sharetheir predicted targets in the IGF signaling. Repression of the IGFsignaling has been shown to inhibit tumorigenesis [Clayton et al. 2011].Thus, these down-regulated miRNAs may assume a role of tumor suppressorsby targeting components of the IGF pathway if their role in regulatingthe IGF signaling is confirmed. The identification of miRNAs asoncogenes or tumor suppressors holds the promise of identifying noveldiagnostic markers or molecular targets for antitumor therapies. Theprediction that the differentially expressed miRNAs have the ability totarget multiple components in one or more pathways makes them potentialmolecular targets for cancer therapy.

Glioblastoma stem cells represent a subpopulation of cancer cells withextraordinary capacities to promote tumor growth, invasion andtherapeutic resistance, making them an ideal target cell population foranti-glioblastoma therapies. However, a major challenge is to definefunctional and molecular features that can distinguish cancer stem cellsfrom normal stem cells in order to develop therapeutic strategies thatspecifically target the tumor population, but leave normal stem cellsintact. Therefore comparing miRNA expression between glioblastoma stemcells and normal neural stem cells is highly relevant in that it maylead to the identification of glioblastoma stem cell-specific miRNAs,thus resulting in the development of novel glioblastoma therapies bytargeting only tumor stem cells. A comparison of the miRNA expressionbetween glioblastoma stem cells from adult glioblastoma patients andnormal neural stem cells from human fetal brains was performed. Fetalbrains were used instead of human adult brains due to theinaccessibility of normal human adult brain tissues containing neuralstem cells. Although differences do exist between embryonic neural stemcells and adult neural stem cells, it has been proposed that embryonicneural stem cells resemble adult neural stem cells in many ways [Ming &Song 2011]. Therefore this comparison will provide useful informationregarding glioblastoma stem cell-specific miRNA expression and provide abasis for strategic targeting glioblastoma stem cells through modulationof tumor stem cell-specific miRNA expression.

miRNAs have been shown to be involved in tumor initiation andprogression, functioning as oncogenes or tumor suppressor[Asadi-Moghaddam et al. 2010; Cheng et al. 2010]. Therefore, modulationof miRNA expression provides great hope for potential cancer therapy[Verissimo et al. 2011]. Furthermore, since each miRNA may have morethan one targets, miRNA-based gene therapy offers the therapeutic appealof targeting multiple gene networks that are controlled by a singlemiRNA [Asadi-Moghaddam et al. 2010]. Strategies for miRNA-based cancertherapy include overexpression of tumor suppressor miRNAs and targetingoncogenic miRNAs using their antagonists. Based on the miRNA signaturethat was identified in glioblastoma stem cells, targeted glioblastomatherapies may be developed by inhibiting the up-regulated miR-10a ormiR-10b function using miR-10 antagonists or overexpressing thedown-regulated miR-124 or miR-874. Of note, 16 miRNAs that wereup-regulated in glioblastoma stem cells (>1.5-fold, as shown in Table2), including miR-10a and miR-10b, are also up-regulated in malignantastrocytomas (glioblastomas and anaplastic astrocytomas) in agenome-wide miRNA expression profiling between malignant astrocytomasand normal brain samples [Rao et al. 2010]. Eleven miRNAs that weredown-regulated in glioblastoma stem cells (>1.5-fold, as shown in Table2), including miR-124, are also down-regulated in malignantastrocytomas.

Moreover, the prognosis of glioblastoma patients remains poor.Biomarkers for this disease are needed for early detection of tumorprogression [Roth et al. 2011]. The miRNA signature that was identifiedherein may be used as biomarkers to differentiate glioblastoma stemcells from normal neural stem cells. Recently, an miRNA signature wasidentified in the peripheral blood of glioblastoma patients [Roth et al.2011]. Interestingly, several of the miRNAs that showed elevatedexpression in the blood samples of glioblastoma patients (vs healthycontrol) also exhibited increased expression in glioblastoma stem cells(vs normal neural stem cells) in the study described herein (Table 2),including miR-424, miR-148a, miR-362-3p, miR-30d, miR-128. These miRNAsmay therefore represent easily accessible biomarkers that can be usedfor diagnostic purposes in glioblastoma patients.

Example 2 miR-874 Acts as a Tumor Suppressor to Target and SuppressGlioblastoma Stem Cells

In the studies described above, an miRNA profiling analysis using bothmicroarray and deep sequencing analyses revealed that one of themiRNAs—miR-874—was significantly down-regulated in glioblastoma stemcells (GSCs) relative to normal stem cells (NSCs). These studies suggestthat this miRNA may function as a tumor suppressor. As such, the role ofmiR-874 in GSC growth was characterized in a study using two GSC celllines derived from WHO grade IV glioblastoma patients, PBT017 andPBT707. A lentiviral expression system was used to deliver miR-874 tothe two GCS cells lines.

Transduction of the GSC lines with miR-874-expressing lentivirus led torobust expression of miR-874 in both types of cells, as shown in FIG. 7A(PBT017 cells) and FIG. 7C (PBT707 cells). The PBT017 and PBT707 cellsoverexpressing miR-874 were cultured for approximately 8-10 days toobserve miR-874's effect on cell growth. A growth curve analysisrevealed that overexpression of miR-874 led to dramatically reduced cellnumbers in both PBT017 and PBT707 glioblastoma stem cell lines, as shownin FIGS. 7B and 7D. These results suggest that miR-874 may act as apotential tumor suppressor miRNA and may be used in accordance with themethods for treating cancers (e.g., glioblastoma) described herein.

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The references, patents and published patent applications listed below,and all references cited in the specification above are herebyincorporated by reference in their entirety, as if fully set forthherein.

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1. A method of treating a cancer comprising: administering atherapeutically effective amount of a pharmaceutical composition to asubject having the cancer, wherein the pharmaceutical compositioncomprises one or more therapeutic agents which target one or more miRNAmolecules that is upregulated in cancer stem cells as compared to normalcells. 2-4. (canceled)
 5. The method of claim 2, wherein the one or moretherapeutic agents includes at least one miRNA inhibitor that inhibitsan upregulated miRNA molecule.
 6. The method of claim 5, wherein theupregulated miRNA is selected from miR-10a, miR-10b, miR-140-3p,miR-140-5p, miR-204, miR-424, miR-34a, miR-193a-3p, miR-455-5p,miR-455-3p, miR-9, miR-10a, miR-148a, miR-488, miR-196a1, miR-182,miR-96, miR-193b, miR-27a, miR-196b, miR-10b, miR-29b2, miR-23a,miR-107, miR-542-3p, miR-93, miR-365a4, miR-450a, miR-100, miR-105,miR-363, miR-105, miR-106b, miR-15b, miR-21, miR-376c, miR-93, miR-99b,miR-155, miR-33a, miR-876-3p, miR-362-3p, miR-25, let-7i, miR-423-3p,miR-34b, miR-16-2, miR-29a, miR-30d, miR-320, miR-181c, miR-128a,miR-21, let-7d, and miR-450b-5p.
 7. The method of claim 1, wherein thecancer is glioblastoma. 8-22. (canceled)
 23. The method of claim 5,wherein the at inhibitor comprises a nucleic acid that is sufficientlycomplementary to the miRNA molecule to hybridize to the miRNA moleculeunder physiological conditions.
 24. The method of claim 1, furthercomprising administering to the subject at least one miRNA that is downregulated in a cancer stem cell as compared to a normal cell or an miRNAexpression vector that overexpresses a downregulated miRNA molecule. 25.The method of claim 24, wherein the downregulated miRNA molecule isselected from miR-371-5p, miR-1245, miR-335, miR-492, miR-874, miR-30b,miR-193a-5p, miR-602, miR-346, miR-663, miR-25, miR-219-5p6, miR-184,miR-135a7, miR-584, miR-665, miR-638, miR-503, miR-628-3p, miR-381,miR-78, miR-92b, miR-149, miR-135b, miR-302d, miR-498, miR-766,miR-1389, miR-623, miR-519c-5p, miR-182, miR-494, miR-129-5p10,miR-513-5p, miR-200b, miR-634, miR-654-5p, miR-518b, miR-658, miR-373,miR-30c-2, miR-130a, miR-557, miR-551a, miR-637, miR-518c, miR-525-5p,miR-596, miR-552, miR-625, miR-183, miR-187, miR-544, miR-891a,miR-519e, miR-933, miR-939, miR-214, miR-671-5p, miR-137, miR-92b,miR-525-3p, miR-19a, and miR-409-5p.